This invention relates to a tissue culture method for the asexual propagation of pine or coniferous trees, said method includes producing multiple adventitious buds from seeds as an essential step.
The current practice of asexual propagation of pine trees through tissue culture requires, as the explant source, embryonic tissue excised from seeds or the use of tissue from young seedlings. From an operational viewpoint, older tissue culture methods, as applied to seed tissues, are unacceptable because of the time and tedious manual labor associated with excising embryonic tissue. Additionally, such methods suffer in that multiple adventitious buds from excised embryonic tissue, as required for asexual propagation, are induced only after a minimum of four weeks.
For example, according to H. E. Sommer, et al., Differentiation of Plantlets in Longleaf Pine (PINUS PALUSTRIS MILL) Tissue Cultured in Vitro, Bot. Gaz., 136, at pp. 196-200 (1970), embryos were dissected from surfaced sterilized seeds of longleaf pine. Thereafter, the embryos were grown in tissue culture media for four to six weeks. Signs of adventitious bud initiation appeared only after four weeks. Similarly, Tsai-Ying Cheng, Adventitious Bud Formation in Culture of Douglas Fir (PSEUDOTSUGA MENZIESII (MIRB) FRANCO), Plant Science Letters, 5, at pp. 97-102 (1975), reports that embryos excised from sterilized decoated seeds of Douglas-fir produced excisable adventitious buds in a tissue culture method after four weeks. These two references are hereby incorporated by reference.
It is known to modify otherwise conventional tissue culture media, comprising standard nutrients, to produce a desired effect. For example, such modification includes adding specified amounts of one or more of the following growth regulators: benzylaminopurine (6-benzylaminopurine), zeatin, 6-.gamma.,.gamma.-dimethylallylaminopurine, kinetin, abscisic acid, indole-3-acetic acid, .gamma.-indolebutyric acid and .gamma.-naphthaleneacetic acid. For example, Asha Mehra-Palta, Richard H. Smeltzer and Ralph L. Mott, "Hormonal control of induced organogenesis," Experiments with excised plant parts of loblolly pine, TAPPI, 61, No. 1, (1978), describe a tissue culture media containing suitable nutrients, modified by the addition of one or more of the following, as growth regulators: benzylaminopurine, zeatin, 6-.gamma.,.gamma.-dimethylallylaminopurine, kinetin and .gamma.-naphthaleneacetic acid. A synergistic effect was attributed to media containing .gamma.-naphtaleneacetic acid, at a low concentration, and one other growth regulator. R. N. Basu et al., "Interaction of Abscisic Acid and Auxins in Rooting of Cuttings, Plant & Cell. Physical., 11, 681-684 (1970), report that abscisic acid at optimum concentrations promoted rooting of PHASEOLUS AUREUS ROXB. and LYCOPERSICON ESCULENTUM MILL. stem cuttings. A synergistic effect of abscisic acid was noted on .gamma.-indolebutyric acid-induced rooting of LYCOPERSICON cuttings. Abscisic acid was suggested as a potential important natural regulator or rooting in cuttings. Arie Altman et al., Promotion of Callus Formation by Abscisic Acid in Citrus Bud Cultures, Plant Physical., 47, 844-846 (1971) report that abscisic acid promoted callus formation in explants of citrus plants. Additionally, benzylaminopurine demonstrated no synergistic effect on abscisic acid-induced callus formation, although other growth regulators produced such an effect. Altman et al, reported culture medium containing benzylaminopurine at a concentration of 218 mg./l and abscisic acid at a concentration of 264 mg./l. Each of these three references are hereby incorporated by reference.